Drug addiction is a chronic relapsing disorder that is characterized by compulsive drug-seeking and -taking behavior even after prolonged periods of abstinence. Relapse prevention is a key treatment challenge because relapse can be triggered by numerous drug-associated conditioned stimuli (CS) and recruits multiple brain circuits. The lateral orbitofrontal cortex (lOFC) and basolateral amygdala (BLA) are two brain regions that are theorized to play a critical role in drug relapse by evaluating the motivational significance of previously drug- paired CSs. This K99 research proposal will examine the importance of monosynaptic connections between the lOFC and BLA in CS-induced goal-directed behaviors. Importantly, the direction of critical information processing will be evaluated for the first time using a sophisticated combination of circuit-specific viral manipulations and optogenetic approaches. Specific Aim 1 will be to examine the role of monosynaptic lOFC-BLA subcircuits in CS-induced cocaine-seeking behavior. To determine whether monosynaptic lOFC to BLA or BLA to lOFC subcircuits are required for CS-induced reinstatement of cocaine-seeking, we will optically inhibit lOFC neurons that project to BLA or BLA neurons that project to lOFC, time-locked with discrete CS presentations during the reinstatement test. Specific Aim 2 will be to examine the role of monosynaptic lOFC-BLA subcircuits in CS-induced food-seeking behavior. In order to determine whether monosynaptic lOFC to BLA or BLA to lOFC sub circuits selectively promote cocaine-seeking behavior, we will explore their involvement in CS-induced natural reward-seeking behavior. The K99 proposal will provide the basis for the R00 research plan and a future R01 proposal. In the R00 phase, Aim 3 will be to explore whether alternate lOFC and BLA afferents are engaged in CS-induced cocaine-seeking behavior. We will use fluorescent retrograde tracers and c-fos immunohistochemistry to identify CS-activated lOFC neurons which send projections to both the BLA and additional brain regions (i.e., dorsomedial prefrontal cortex, nucleus accumbens, ventral hippocampus, Medio dorsal thalamus) that may also be critical for CS-induced cocaine- seeking behavior (and vice versa for BLA neurons). Utilizing the technical approach optimized in Aims 1, R00 follow-up studies and a future R01 will establish the functional significance of these collaterals in CS-induced reinstatement. In summary, the proposed studies will elucidate the significance of lOFC-BLA sub circuits in CS-induced motivated behavior. Understanding the functional connectivity of these brain regions as well as the larger relapse circuitry may enhance our ability to identify and reverse pathological neuroadaptations that confer extreme cue reactivity and treatment resistance in cocaine users. The corresponding research training plan will be fundamental for the PI's successful transition to an independent researcher in the addiction field by allowing her to acquire cutting-edge research tools, including in vivo viral manipulation, optogenetic procedures, a sophisticated rodent model of drug-relapse, as well as experience in research management.